ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.     

A computerized spectrophotometric instrumental system to determine the "vertical velocity" of sperm cells: a novel concept.

BACKGROUND: The presently available cell motility-analyzers measure primarily the "horizontal" velocity and there is no instrument available for "vertical" velocity measurement. This development was based on the turbidimetric method of sperm motility analysis. METHODS: Sperm was layered at the bottom of the cuvette containing buffer solution and exposed to the spectrophotometric light path at different heights to track the vertically moving sperms. The vertical movement was materialized with the development of an electromechanical up-down movement devise for the cuvette accomplished with the help of a cuvette holder-stepper motor-computer assembly. The entire system was controlled by the necessary motion control, data acquisition, and data processing software developed for cuvette movement and data analysis. RESULTS: Using goat sperm as the model a unique computer-based spectrophotometric system has been developed for the first time to determine the average "vertical" velocity of motile cells. CONCLUSIONS: Undertaking upward movement against gravity is much tougher as compared with horizontal movement. Consequently average vertical velocity is expected to be a much better identifying parameter for assessing semen and other motile cell quality. The novel instrumental system developed by us has thus the potential for immense application in human infertility clinics, animal-breeding centres, centres for conservation of endangered species, and also for research work on vertical velocity of spermatozoa and other motile cells, such as bacteria, protozoa, etc.     

Cobalt(III) complexes of some aromatic thiohydrazides - Synthesis, characterization and structure

[Co(NH₃)₅Cl]Cl₂ forms neutral 1:3 complex by reaction with aromatic thiohydrazides, i.e. thiobenzhydrazide, o-hydroxythiobenzhydrazide, thiophen-2-thiohydrazide and furan-2-thiohydrazide. All these complexes are diamagnetic and have been characterized by elemental analysis and combination of spectroscopic methods. Cyclic voltammometry of the complexes shows irreversible metal centered and ligand centered electron transfer reactions. One complex, tris-o-hydroxythiobenzhydrazidocobalt(III), has been crystallized from DMSO solution to produce solvated crystals and its structure has been established by X-ray crystallography. Cobalt(III) ion is linked through three hydrazinic nitrogen and three sulfur atoms of three identical deprotonated ligand molecules in a distorted octahedral environment. Involvement of -OH group in intramolecular and intermolecular hydrogen bonding is crucial for crystal formation.     

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.     

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65–75 years) were incubated with aggrelyte-2 (500 μM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%–30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC–MS/MS results showed N^ε-acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.     

Temperature extremes, density dependence, and southern pine beetle (Coleoptera: Curculionidae) population dynamics in east Texas

Previous studies of the southern pine beetle, Dendroctonus frontalis Zimm., established that its population in east Texas responds to a delayed density-dependent process, whereas no clear role of climate has been determined. We tested two biological hypotheses for the influence of extreme temperatures on annual southern pine beetle population growth in the context of four alternative hypotheses for density-dependent population regulation. The significance of climate variables and their interaction with population regulation depended on the model of density dependence. The best model included both direct and delayed density dependence of a cubic rather than linear form. Population growth declined with the number of days exceeding 32 degrees C, temperatures previously reported to reduce brood survival. Density dependence also changed with the number of hot days. Growth was highest in years with average minimum winter temperatures. Severely cold winters may reduce survival, whereas warm winters may reduce the efficiency of spring infestation formation. Whereas most previous studies have incorporated climate as an additive effect on growth, we found that the form of delayed density dependence changed with the number of days >32 degrees C. The interaction between temperature and regulation, a potentially common phenomenon in ecology, may explain why southern pine beetle outbreaks do not occur at perfectly regular intervals. Factors other than climate, such as forest management and direct suppression, may have contributed significantly to the timing, severity, and eventual cessation of outbreaks since the mid-1950s.     

Dendritic cell-based immunotherapy combined with antimony-based chemotherapy cures established murine visceral leishmaniasis

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4(+) T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.     

Regional variations in fibre growth dynamics of myotomal and caudal fin muscles in relation to body size of a freshwater teleost, Barbus sarana (Cuv. & Val.)

The growth of red fibres in anterior and middle myotomal regions of B. sarana was mainly by hyperplasia in smaller size classes. In higher size classes, growth by hyperplasia was greater in posterior myotomal region compared to the other two myotomal regions. The growth of pink fibres in anterior myotomal regions was mainly by hypertrophy. The middle and posterior myotomal regions showed fibre growth by hyperplasia. The growth dynamics of white fibres revealed more or less similar pattern in all three myotomal regions against the somatic development. White fibres grew by hyperplasia up to 8 cm F.L. size classes and thereafter by hypertrophy. However, in > 12 cm F.L. size classes, the mean diameter of white fibres did not increase significantly. Similar pattern of growth was found in the white fibres of caudal fin muscle. It is interesting to note that the hyperplasia was mostly completed in the white fibres of the smallest fish studies, whereas, it continued to quite larger fish size in red and pink fibres. Thus, hyperplasia and hypertrophy may be responsible for growth in all fibre types in all myotomal regions in relation to somatic development in this small and medium growing species.